The terpenoids, also called isoprenoids, constitute the largest family of natural products with over 22,000 individual compounds of this class having been described. The triterpenes or terpenoids (hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, polyprenols, and the like) play diverse functional roles in plants as hormones, photosynthetic pigments, electron carriers, mediators of polysaccharide assembly, and structural components of membranes. The majority of plant terpenoids are found in resins, latex, waxes, and oils.
Triterpenoids are of relevance to a variety of plant characteristics, including palatability to animals, and resistance to pathogens and predators. Triterpenes are mostly stored in plant roots as their glycosides, saponins (see Price K. R. et al, 1987, CRC Crit. Rev. Food Sci. Nutr. 26:27-133). Thus, for example, mutants of the diploid oat species, Avena strigosa, which lack the major oat root saponin, avenacin A-1 (so called saponin-deficient or “sad” mutants) have been shown to have compromised disease resistance (Papadopoulou K. et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:12923-12928). These mutants have increased susceptibility to a number of different root-infecting fungi, including Gaeumannomyces graminis var. tritici, which is normally non-pathogenic to oats. Genetic analysis suggests that increased disease susceptibility and reduced avenacin content are causally related. Furthermore, a sad mutant which produces reduced avenacin levels (around 15% of that of the wild type) gives only limited disease symptoms when inoculated with G. graminis var. tritici in comparison to other mutants which lack avenacins completely, providing a further link between avenacin content and disease resistance.
Triterpenoid saponins are synthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give pentacyclic triterpenoids, primarily oleanane (β-amyrin) or dammarane skeletons. The triterpenoid backbone then undergoes various modifications (oxidation, substitution, and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases, and other enzymes. In general very little is known about the enzymes and biochemical pathways involved in saponin biosynthesis. The genetic machinery required for the elaboration of this important family of plant secondary metabolites is as yet largely uncharacterized, despite the considerable commercial interest in this important group of natural products. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. However, the first dedicated step in saponin biosynthesis is now understood to be carried out by the oxidosqualene cyclase β-amyrin synthase, which has recently been cloned and characterized (Haralampidis K. et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98:13431-13436).
Many of the primary modifications to β-amyrin indicated in FIG. 1, which compares the structures of β-amyrin and avenacin A-1, are likely to be mediated by cytochrome P450 monooxygenases. These include oxidation at C16, C21, C30, or C23, and epoxidation at C12, C13. Besides their involvement in saponin biosynthesis, cytochrome P450 monooxygenases are involved in the biosynthesis of a multitude of other compounds, as described in (Nelson D. R., 1999, Arch. Biochem. Biophys. 369:1-10). While some single cytochrome P450 monooxygenase enzymes can metabolize multiple substrates, many of these enzymes are highly substrate specific. For example, in maize four P450s (BX2-5) sharing 45-60% amino acid identity belonging to the CYP71C family carry out successive hydroxylation events in the conversion of indole to the cyclic hydroxamic acid 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), each enzyme catalyzing predominantly only one reaction in the pathway. Available P450 structures show that the overall P450 structural fold is preserved during evolution from bacteria through plants and mammals. At the same time there are variable regions that appear to be associated with recognition and binding of structurally diverse substrates and redox partners.
The CYP51 (sterol 14α-demethylase) family is an essential enzyme in sterol biosynthesis and is the only P450 family that serves the same function in different biological kingdoms (Lepesheva G. I. et al., 2003, Biochemistry 42:9091-9101; Kelly S. L. et al., 2001, Biochem. Soc. Trans. 29:122-128). CYP51 enzymes catalyze the oxidative removal of the 14α-methyl group from lanosterol and 24-methylene-24,25-dihydrolanosterol in yeast and fungi, from obtusifoliol in plants and from 24,25-dihydrolanosterol in mammals. The products of action of sterol 14α-demethylases are Δ14,15-desaturated intermediates in ergosterol (fungi), phytosterol (plants) and cholesterol (animals) biosynthesis. The reaction includes three steps of successive conversion of the 14α-methyl group to 14α-hydroxymethyl, 14α-carboaldehyde, and 14α-formyl intermediates followed by elimination of formic acid with concomitant introduction of the Δ14,15 double bond into the sterol core. CYP51s are targets for antifungal and cholesterol-lowering drugs.
The present invention describes polynucleotides encoding novel CYP51s, one of which modifies β-amyrin or a β-amyrin derivative. Identification of the genes encoding enzymes responsible for modification of β-amyrin or β-amyrin derivatives in a variety of crops will allow the manipulation of the same. Manipulation of the β-amyrin pathway will result in changes in the levels or structures of the saponins. A decrease in saponin production will result in an enhancement of plant resistance to pests. Foods originating from plants having an increased level of triterpenes are thought to have a cholesterol lowering effect while decreased triterpenes are believed to result in better tasting foods. Thus, transgenic plants having altered levels of triterpenes may be resistant to pests and foods prepared with seeds having altered levels or structures of saponins will have increased nutritional value or better flavor.